Cloning Vectors

Baculoviral Expression System

Gives overall schematic for making proteins in baculoviral system. Start from upper left. There is a plasmid where inside is a region to insert DNA with a promoter and expression vector. Put DNA in there behind promoter and transform DH-10 cells (modified for use), the donor plasmid, bacmid is a phagemid with duality between single/double strands. The cell then can switch our inserted DNA from plasmid into the bacmid. The process is used over and over "homologous recombination" is the name. The DNA between the flanking regions are prone to being switched. In the E. Coli, now, the final bacteria, the bacmid has the DNA and is within the E. Coli. Next, we extract bacteria and bacmids and they are inserted into insect cells that have these viruses within. In the cells, they will now be inserted into the active viral particles. Virus particles have the DNA of interest and then these are taken to infect insects and these cells are identified for the DNA of interest. From working with DNA of interest in plasmid to switching them from a different vector, into the baculoviral system and then into the insects.

Plasmid

MOST used cloning vector; ds, closed circular DNA; pBR322 has 15-20 copies and pUC 500-700 copies number of copies varies; gene encoded by plasma include drug resistance

Yeast Artificial Chromosome

YAC; synthetic chromosome that are propogated in yeast; has selectable marker, telomeric DNA and centromere for cloning LARGE DNA pieces TELOMERE keeps artificial complex from being consumed; makes it look like a chromosome

Host Cells

Yeast, Mammalian cells (COS cells), Xenopus oocytes, insect cells E. coli

Single Strand Propagation

co-infect E. coli with both phagemid and helper bacteriophage; ssDNA in packed into phage particle to infect other cells

Genomic DNA

contains ENTIRE gene sequence

Complementary DNA (cDNA)

contains ONLY protein coding region of gene

Phagemid

hybrid of plasmid/filamentous bacteriophage; plasmid with dsDNA using ori or bacteriophage using f1 ori

Baculovirus

large rod-shaped virus to infect insect cells; contains circular dsDNA for easy insertion and for expression of glycosylated proteins

Cloning Vector

modifies DNA sequences within unique DNA segment, make multiple copies of DNA/RNA of specific sequence, store DNA in construct

Cosmid

originated as plasmid; cos sequences from bacteriophage lamda inserted intno plasmid. sticky ends manipulated as bacteriophage but expressed as plasmid by host cell

Bacteriophage

ssDNA vectors, closed circular structure, infects E. coli, contains selectable marker of B-galactosidase; turns blue

Expression Vector Expression Vector Elements

synthesizes protein coded by cloned DNA sequence contains these elements: Promoter/Enhancer Elements (as inducible promoter), Ribosomal binding site, Start Codon and strong termination codon